This proposal is focused on developing our understanding of class 1 myosins: ubiquitously expressed monomeric, membrane binding, actin-based motors that participate in diverse cellular functions, including organelle trafficking, transcription, host defense, cell motility, and mechano-sensation. Our studies of myosin-1 are centered on the 'brush border', a tightly packed array of microvilli that extends from the apical surface of many transporting epithelial cells types. This organelle is home to a number of myosin superfamily members, with the most abundant being myosin-1a (Myo1a), one of eight vertebrate class 1 myosins. Our laboratory has leveraged a unique combination of cell biological and biophysical approaches to discover that: (i) Myo1a contributes to membrane-cytoskeleton adhesion, which is critical for maintaining normal brush border structure, and (ii) Myo1a powers the release of membrane vesicles enriched in host defense machinery from microvillar tips into the intestinal lumen. The physiological significance of Myo1a function is also underscored by recent studies showing that mutations in this motor are linked to colorectal tumor formation in humans. While we have made substantial progress toward elucidating the biological roles of Myo1a, the molecular properties, interactions, and events that govern the function of this and other myosins-1 remain poorly characterized. The goal of this proposal is to develop our understanding of the fundamental biochemical and biophysical properties that enable Myo1a to contribute to brush border function. To this end, Aim 1 will examine the unitary properties of single Myo1a molecules interacting with the plasma membrane of live cells and supported bilayers in vitro, Aim 2 will examine the force generating potential of Myo1a bound to supported bilayers, and Aim 3 will investigate the role of force sensing in the regulation of Myo1a dynamics and function. Because defects in brush border formation and maintenance are at the core of numerous diseases that pose significant threats to human health, developing insight on the molecular mechanisms that govern Myo1a behavior will provide information that may ultimately be used in the development of therapeutics aimed at repairing malformed or damaged brush borders.